Peptides need to be in highly pure form for analytical purposes or where biologicals are to be used as medicaments. Both synthetic and recombinant peptides generally require at least some purification as part of the manufacture process before they can be used. When attempting to separate peptides in a complex mixture some are so different from the others that purification methods based on protein size, physico-chemical properties and binding affinity are able to separate them. However, the vast majority of proteins are sufficiently similar that special measures are needed to separate mixtures. One such measure involves attaching an affinity ‘tag’ to a protein to give the protein a binding affinity it would not ordinarily have. If the tag makes the protein unique in the mixture, the affinity can be used to separate the protein from the others.
The inherent affinity of some amino acids, such as histidine (His), to metal ions has been utilised in the purification of some peptides and proteins. One technique involves adding a poly-His (E. Hendan and J. Porath, J. Chromatography, 1985, 323, 255-264) sequence to the terminus of the peptide or protein sequence of interest, and using the affinity of the appendage for nickel ions to selectively bind the desired product to a surface. Although effective, the poly-His tag remains a permanent feature of the parent sequence, which may affect protein folding. Moreover, the extra synthetic steps to add the tag may reduce overall yield of material.
A cleavable poly-His tag has been reported (Servion et al, EP 0827966), in which a methionine residue is inserted between the parent peptide sequence and the histidine residues. Cleavage at methionine residues is achieved selectively by treatment with cyanogen bromide which is a highly toxic reagent. Furthermore, methionine residues in the protein sequence used in the oxidised form to prevent undesired cyanogen bromide cleavage must be reduced again to obtain the native sequence, increasing complexity of the synthesis.
There are few examples of chemical tags which have been used to enhance the properties of chemically synthesised peptides and proteins. Ramage et al (R. Ramage and G. Raphy, Tetrahedron Lett., 1992, 33, 385-388) used Tbfmoc as a hydrophobic analogue to Fmoc for the purification of peptides and proteins. Tbfmoc was found to be extremely hydrophobic, and has sufficient effect on the elution of peptides and proteins with the tag appended, to assist in preparative HPLC purification. However, the Tbfmoc group was found to have such a high affinity for hydrophobic surfaces that undesired binding to surfaces was often unavoidable. Furthermore, Tbfmoc has an unfavourable effect on the solubility of peptides and proteins in aqueous systems, often resulting in difficulties during purification.
Tags that covalently bind to a functionalized solid support have been used to purify both synthetic and recombinant proteins (M. Villain, J. Vizzavona, and K. Rose, Chemistry & Biology, 2001, 8, 673-679; J Vizzavona, M. Villain, and K. Rose, Tetrahedron Lett, 2002, 8693-8696). Initially, the method was applicable only to proteins with N-terminal cysteine or threonine, but the method was tailored to be suitable for all N-terminal amino acids (J Vizzavona, M. Villain, and K. Rose, Tetrahedron Lett, 2002, 8693-8696). However, obtaining the chemical tag requires a lengthy synthesis, and where removal is required the sodium periodate cleavage step can damage the peptide or protein.
Non amino acid ‘chemical’ affinity tags that can be reversibly attached to peptides are disclosed in EP1628998. These tags allow the peptide to be purified by passage through a column of immobilised metal ions. Synthesis of tagged amino acids by the method taught is a multistage reaction with several difficult steps and chemical stability of the tagged peptides is suboptimal.
It would be useful if there were a compound that could be used as a tag for the affinity tagging of peptides and proteins that could address these shortcomings of the art. In particular it would be useful if there were a group of easy-to-synthesise, robust tagging molecules that obtained these advantages without any detraction from the ease in which the coupling chemistry could be carried out. There also remains a need for chemical tags which can be used for purification of a peptide and which may be efficiently and easily cleaved from the peptide whilst minimising damage to the peptide itself.
It is an object of the present invention to address any and all of these aforementioned needs in the art or at the very least to provide the public with a useful alternative to those art known methods and compounds.